非洲猪瘟病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒多重TaqMan荧光定量RT-PCR检测方法的建立

发布时间：2018-10-09 17:11

【摘要】：为建立同时检测非洲猪瘟病毒(ASFV)、猪瘟病毒(CSFV)和高致病性猪繁殖与呼吸道综合征病毒(HP-PRRSV)的方法,本研究根据ASFV CP530R基因、CSFV 5'UTR基因和HP-PRRSV NSP2基因,分别设计了3对特异性引物和Taq Man水解探针,建立了同时检测这3种病毒的多重荧光定量RT-PCR方法,并对其反应条件进行优化。结果表明,该方法仅对ASFV、CSFV和HP-PRRSV呈现特异性扩增,不与伪狂犬病毒、猪细小病毒和猪圆环病毒2型的DNA以及猪流行腹泻病毒和猪流感病毒的c DNA发生交叉反应;该方法对ASFV、CSFV和HP-PRRSV的最低检出量分别为61拷贝/μL、11拷贝/μL和41拷贝/μL;组内和组间重复性试验的Ct值变异系数均小于2.5%,具有良好的重现性。用该方法对276份临床样品进行ASFV、CSFV和HP-PRRSV的检测,所有样品的ASFV检测结果均为阴性,CSFV检测单阳性样品6份,HP-PRRSV检测单阳性样品22份,CSFV和HP-PRRSV检测双阳性样品4份。本研究建立的方法为临床样品中ASFV、CSFV和HP-PRRSV的同时检测提供了一种快速、敏感和特异的技术手段。
[Abstract]:In order to establish a method for simultaneous detection of African classical swine fever virus (ASFV),) (CSFV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), the present study was based on ASFV CP530R gene, CSFV 5'UTR gene and HP-PRRSV NSP2 gene. Three pairs of specific primers and Taq Man hydrolyzed probes were designed, and a multiplex fluorescence quantitative RT-PCR method was established for simultaneous detection of the three viruses, and the reaction conditions were optimized. The results showed that the method was only specific for ASFV,CSFV and HP-PRRSV, and did not cross react with pseudorabies virus, porcine parvovirus and porcine circovirus type 2 DNA and porcine epidemic diarrhea virus and swine influenza virus c DNA. The minimum detectable quantities of ASFV,CSFV and HP-PRRSV were 61 copies / 渭 L 11 copies / 渭 L and 41 copies / 渭 L, respectively, and the coefficients of variation of Ct values in intra-group and inter-group reproducibility tests were less than 2.5 copies / 渭 L, respectively, with good reproducibility. The method was used to detect ASFV,CSFV and HP-PRRSV in 276 clinical samples. The results of ASFV detection in all samples were negative. Six samples were positive for HP-PRRSV. 22 samples were positive for HP-PRRSV and four samples were double positive for HP-PRRSV. This method provides a rapid, sensitive and specific technique for simultaneous detection of ASFV,CSFV and HP-PRRSV in clinical samples.
【作者单位】：天津出入境检验检疫局动植物与食品检测中心;
【基金】：天津市科技支撑计划项目(13ZCZDNCO1300)
【分类号】：S852.651

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